A comprehensive analysis of chemical and biological pollutants (natural and anthropogenic origin) of soil and dandelion (Taraxacum officinale) samples

A range of analytical methods (GC-MS, LC-MS, voltammetry, microbiological and microscopic techniques, PCR) was used to assay a range of potential chemical and biological contaminants in soil and dandelion samples. The results provide the first comprehensive safety analysis of dandelion as a herbal product. Samples were collected from three different sites in Poland where the local population collects dandelion plants for their own consumption: Rudenka (a mountain meadow in the European Ecological Network of Natura 2000 protection area, free of agrotechnical treatments for over 30 years), Warszawa 1 (dense single-family housing with heavy traffic), and Warszawa 2 (recreation area with heavy traffic near a coal-fired heat and power plant). The assays of heavy metals and other chemical pollutants (PAHs, PCBs, dioxins, pesticides, mycotoxins) confirm that all collected soil and dandelion samples were chemically pure; however, 95 species of pathogenic bacteria were detected, including “carnivorous” Vibrio vulnificus, zoonotic Pasteurella pneumotropica, Pasteurella canis, Staphylococcus pseudintermedius, Staphylococcus lentus and Francisella tularensis as well as 14 species of pathogenic fungi and one protozoan parasite (Giardia intestinalis). The discovery of septicemia agents V. vulnificus, Fusobacterium mortiferum and Rahnella aquatilis in the soil surrounding dandelion roots and in the flowers, G. intestinalis in dandelion leaves and roots samples, all collected in Warsaw, is highly disturbing. This finding underlines the need for increased caution when collecting dandelion in densely populated areas with a large population of pets. Thorough washing of the harvested plants is necessary before using them for consumption, especially in the case of making salads from fresh dandelion leaves, which is becoming increasingly popular among people leading healthy and an environmentally friendly lifestyle.


Review Comments to the Author
Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: A comprehensive analysis of chemical and biological pollutants (natural and anthropogenic origin) of soil and dandelion (Taraxacum officinale) samples -pathogenic microorganisms and parasites pose a greater threat Although the title and purpose of this manuscript is very interesting, I think that in some cases the correct scientific method was not used. Laboratory methods used for microorganisms are not very clear. Meanwhile, organisms like Francisella grow in specific medium and microaerophilic conditions.
Answer: It is difficult to respond to such a general comment because Reviewer #1 did not mention any specific methodological errors that he believed we had made. In this work, very diverse methods were used, from voltammetric determinations of heavy metals through gas and liquid chromatography coupled with mass spectroscopy (GC-MS and LC-MS), microbiological methods and PCR. All these methods are described in detail in the Materials and Methods section. We expect Reviewer #1 to present specific objections to the methods used by us.
In microbiological studies, we used classical inoculation methods on commercially available media: Columbia Agar plates with 5% sheep blood (CASB) and Sabouraud Glucose Selective Agar plates (SGSA), both from Becton Dickinson. Half of the inoculated CASB plates were kept aerobically, while the second half was kept anaerobically using the anaerobic jar (Roth) and AnaeroGen ™ sachets (Thermo Scientific). All CASB plates were incubated at 37 o C for 24 hours. Following this, the number of colony forming units (CFU) was determined using a SCAN 500 automatic bacterial colony counter (Interscience). The bacteria were Gram stained (Gram Stain Kit, Thermo Scientific) and examined under a microscope to distinguish grampositive and gram-negative colonies and bacterial morphology as this was a prerequisite for strain identification using an automated Vitek-2 system (Biomerieux). Homogeneous bacterial colonies were applied to correctly-selected Vitek-2 disposable cards. The modern, Vitek-2 automated microbiology system is validated and widely used in clinical microbiology to identify various bacterial strains in diagnostic laboratories and hospitals around the world (https://www.biomerieux-usa.com/vitek-2; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC254354/ ). The composition of the growing media used in the Vitek-2 system cards dedicated various groups of microorganisms is a manufacturer's secret.
Francisella tularensis might be grown in defined media such as Chamberlains or in nonselective media such as Mueller-Hinton broth and a modified Brain Heart Infusion (BHI) broth enhances the growth of this microbe (Morris BJ, Buse HY, Adcock NJ, Rice EW. A novel broth medium for enhanced growth of Francisella tularensis. Lett Appl Microbiol. 2017 Jun;64(6):394-400. doi: 10.1111/lam.12725). We do not know which medium is used in the Biomerieux cards for Francisella tularensis identification (manufacturer secret).
Reviewer #2: The present work is a good effort to detect chemical and biological contaminations in soil and dandelion samples using a wide range of analytical methods (GC-MS, LC-MS, voltammetry, 505 microbiological and microscopic techniques, PCR). I agree with its publication. However, herewith I have few suggestions for its improvement • Revise the title for clarity Answer: Revised title: "A comprehensive analysis of chemical and biological pollutants (natural and anthropogenic origin) of soil and dandelion (Taraxacum officinale) samples." • Make abstract and introduction short Answer: The abstract is only 268 words long and is written in a very concise manner. Attempts to shorten it result in a loss of clarity. The Introduction has been shortened in such a way as not to lose important information and clarity of the message, as well as the justification why the research described in this work was carried out.